Use of a polypeptide as cellular receptor for adenoviruses

ABSTRACT

The subject of the present invention is the use of a polypeptide comprising at least 6 continuous amino acids of the sequence as shown in the sequence identifiers 1 to 5 as cellular receptor and/or coreceptor for adenoviruses. It also relates to the use of a cell capable of expressing such a polypeptide as well as that of a ligand capable of influencing the attachment of an adenovirus to a host cell and/or its entry into the said host cell. Finally, it also relates to a method for selecting or identifying a cellular receptor for a virus or the part of a viral protein which determines the attachment of the virus to its cellular receptor as well as to the use of a bifunctional ligand to target an adenovirus to a host cell carrying, at its surface, a surface protein other than the natural cellular receptor for the said adenovirus.

[0001] The subject of the present invention is the use of all or part ofan antigen of the class I major histocompatibility complex and/or of atype III module of fibronectin to allow or facilitate the attachment ofan adenovirus onto a host cell and/or its entry into the latter. Theinvention also relates to the use of a ligand capable of modulating theinfectivity of an adenovirus toward a host cell, mediated by either ofthe polypeptides mentioned above. Finally, the invention relates to abiopanning method for identifying or selecting a cellular receptor foran adenovirus or one of these ligands, in particular of viral origin.

[0002] Adenoviruses are DNA viruses with a broad host spectrum. Theyhave been detected in numerous animal species and can infect variouscell types. Numerous serotypes have been characterized within eachspecies which exhibit a genomic organization and an infectious cyclewhich are comparable. In general, the adenoviral genome consists of adouble-stranded linear DNA molecule of about 36 kb containing the genesencoding the viral proteins and, at its ends, two inverted repeats(designated ITRs) which are involved in replication and theencapsidation region.

[0003] Adenoviruses replicate in the nuclei of the cells infected. Theinfectious cycle occurs in two stages. The early phase precedes theinitiation of replication and makes it possible to produce the earlyproteins regulating the replication and the transcription of the viralDNA. These stages are followed by the late phase during which thestructural proteins which constitute the viral particles aresynthesized. The assembling of the new virions takes place in thenucleus. In the first instance, the viral proteins assemble so as toform empty capsids having an icosahedral structure, into which theadenoviral DNA is encapsidated. The viral particles are released and arecapable of infecting other permissive cells. In this regard, the fiberand the penton base which are present at the surface of the capsids playa critical role in the cellular attachment of the virions and theirinternalization.

[0004] The adenovirus binds to the surface of permissive cells throughthe intermediacy of the trimeric fiber and a cellular receptor which hasso far not been identified. Next, the particle is internalized byendocytosis through the binding of the penton base to the cellularintegrins α_(v)β₃ and α_(v)β₅ (Belin and Boulanger, 1993, J. Gen. Virol.74, 1485-1497; Mathias et al., 1994, J. Virol. 68, 6811-6814; Nemerow etal., 1994, Trends Cell. Biol. 4, 52-55; Wickham et al., 1993, Cell 73,309-319; Wickham et al., 1994, J. Cell Biol. 127, 257-264). The Ad2fiber comprises 580 amino acids (aa) whose sequence is disclosed inHerissé et al. (1981, Nucleic Acid Res. 9, 4023-4042). That of Ad5 has582 amino acids (Chroboczek and Jacrot, 1987, Virology 161, 549-554).Its molecular mass is 62 kDa, but the native fiber behaves like a160-180 kDa molecule, confirming its assembly in the form of a trimer.

[0005] The fiber is composed of 3 domains (Chroboczek et al., 1995,Current Top. Microbiol. Immunol. 199, 165-200):

[0006] (1) At the N-terminus, the “tail”, which is highly conserved fromone serotype to another, interacts with the penton base and ensures theanchorage of the molecule in the capsid.

[0007] (2) The “stem” is a structure in the form of a rod of variablelength depending on the serotypes. For example, the stem of the Ad5fiber contains 22 repeats of a motif of 15 residues which could adopt aβ sheet conformation. The number of these repeats differs from oneserotype to another, which explains the variations in length.

[0008] (3) Finally, at the distal end of the stem, the “head” orterminal sphericle is a globular structure containing trimerizationsignals (Hong and Engler, 1996, J. Virol. 70, 7071-7078; Novelli andBoulanger, 1991, J. Biol. Chem. 266, 9299-9303; Novelli and Boulanger,1991, Virology 185, 365-376). Most of the experimental data show that itis the head domain which is responsible for the binding to permissivecells (Krasnykh et al., 1996, J. Virol. 70, 6839-6846).

[0009] (4) The complexity of the adenoviral attachment suggests that itcould be serotype-dependent and that several cellular proteins couldparticipate in it. As regards Ad2, Hong and Boulanger (1995, EMBO J. 14,4714-4727) have identified a number of peptide motifs found in severalcellular surface proteins which are capable of interacting with thecapsid proteins (penton base and fiber), in particular the type III 5and 14 modules of human fibronectin. The authers proceeded byimmobilizing, on an inert support, penton base or fiber (ligand) withwhich they reacted a library of phages expressing random hexapeptides(designated phagotopes). The phages adsorbed, which in theory expressphagotopes interacting with a motif carried by the adenoviral protein,are then eluted either conventionally at acidic pH or by competitionwith the other nonimmobilized capsid partner (eluent). However, thecellular receptor for adenoviruses and the region of the head preciselyinvolved in the binding to the receptor have so far not yet been clearlyidentified.

[0010] A new technique of “biopanning” has now been carried out in whichthe immobilized ligand consists of the head domain of the Ad5 fiber andthe eluent consists of a neutralizing antibody directed against thelatter and two classes of phagotopes isolated depending on the antibodyused. The first corresponds to a conserved sequence within the α-2domain of the antigens of the class I major histocompatibility complex(α-2 MHC-I) and the second to a sequence found in the III modules ofhuman fibronectin (FNIII). The data presented in the examples whichfollow support the hypothesis that the α-2 MHC-I constitutes the primaryreceptor for the serotype C adenoviruses and confirm the participationof the FNIIIs as coreceptor or cofactor. The regions of these tworeceptors and of the fiber which interact with each other have also beenidentified. In addition, an antagonist peptide has been generated whichreproduces the motif of the α-2 MHC-I domain which neutralizes theattachment of adenoviruses and an agonist peptide reproducing the FNIIImotifs which stimulates attachment.

[0011] Accordingly, the subject of the present invention is the use of apolypeptide comprising an amino acid sequence homologous or identical toat least 6 continuous amino acids of the sequence as shown:

[0012] (a) in SEQ ID NO: 1 starting with the leucine residue at position1 and ending with the glutamine residue at position 25,

[0013] (b) in SEQ ID NO: 2 starting with the asparagine residue atposition 1 and ending with the asparagine residue at position 26,

[0014] (c) in SEQ ID NO: 3 starting with the valine residue at position1 and ending with the asparagine residue at position 25,

[0015] (d) in SEQ ID NO: 4 starting with the serine residue at position1 and ending with the arginine residue at position 25, and/or

[0016] (e) in SEQ ID NO: 5 starting with the asparagine residue atposition 1 and ending with the serine residue at position 25;

[0017] to allow or facilitate the attachment of an adenovirus to a hostcell and/or the entry of the said adenovirus into the said host cell.

[0018] For the purposes of the present invention, “polypeptide” isunderstood to mean any molecule consisting of a succession of at least6, and preferably of at least 8, amino acids. The term polypeptidecomprises both peptide molecules of short length (from 6 to a few tensof residues) and molecules which are longer (up to several hundreds ofresidues), provided, however, the envisaged use is allowed. It isspecified that a polypeptide in use within the framework of the presentinvention may be derived from a native polypeptide as found in nature,in particular in humans, or a portion thereof. It may also be a chimeraand comprise additional residues of any origin fused at the N- and/orC-terminus and/or inserted so as to form an open reading frame. It isalso possible to use a mutant obtained by mutation, deletion, insertionand/or substitution of one or more amino acids relative to the sequencesdisclosed in the sequence identifiers (SEQ ID).

[0019] A preferred polypeptide within the framework of the presentinvention comprises, in addition, appropriate elements to ensure itsanchorage in a cell membrane or its presentation at the surface of acell. Such elements are known to persons skilled in the art. As a guide,there may be mentioned the presence of a signal peptide generallyassociated at the N-terminal position and of a transmembrane regionexhibiting a high degree of hydrophobicity. However, use may also bemade of other techniques, for example chemical techniques, to anchor orbind a polypeptide to a membrane or a cell surface.

[0020] “Homologous amino acid sequence” is understood to mean a sequencehaving a degree of homology of at least 70%, advantageously of at least80%, preferably of at least 90% with at least 6 continuous amino acidsof one of the sequences mentioned. The term identical refers to 100%homology. Persons skilled in the art know the general rules which makeit possible to calculate the degree of homology between two sequences.The procedure is generally carried out by aligning sequences possiblywith the aid of specialist computer programs. It may be necessary toartificially introduce vacant positions. Once the optimum alignment hasbeen achieved, the degree of homology is established by counting all thepositions in which the amino acids of the two sequences are found to beidentical, relative to the total number of positions.

[0021] “Attachment of an adenovirus to a host cell” is understood tomean the binding of the viral particle to the cell. “Entry of anadenovirus into a host cell” denotes the penetration of the virus intothe host cell. The attachment and/or the entry are preferably mediated,at least in part, by the polypeptide(s) in use within the framework ofthe present invention by interaction wish the adenoviral capsid. Ofcourse other polypeptide or nonpolypeptide molecules may alsoparticipate in these processes which are recognized in the art to becomplex and multifactorial. They can be evaluated by any prior arttechnique, such as those described below using a permissive cell lineand particles which are radioactively labeled or which express areporter gene, for example the luciferase gene. At 0° C., only theattachment can take place, the viral penetration requiring a temperatureof 37° C.

[0022] For the purposes of the present invention, an adenovirus may beof human or animal (canine, avian, bovine and the like) or hybrid origincomprising genome fragments. These viruses and their genome aredescribed in the literature (see for example Graham and Prevec, Methodsin Molecular Biology, Vol. 7; Gene Transfer and Expression Protocols;Ed: E. J. Murray, 1991, The Human Press Inc., Clinton, N.J.). Areplication-defective recombinant adenovirus expressing in particular agene of therapeutic interest is preferred. Advantageously, theadenoviral genome is modified by deletion or mutation of sequencesessential for replication and, in particular, contained in the E1, E2,E4 and/or L1-L5 regions (see for example international application WO94/28152).

[0023] According to a first variant, the subject of the presentinvention is the use of a polypeptide comprising an amino acid sequencehomologous or identical to at least 6 continuous amino acids of thesequence as shown in SEQ ID NO: 1 starting with the leucine residue atposition 1 and ending with the glutamine residue at position 25.

[0024] Advantageously, a polypeptide in use within the framework of thepresent invention comprises an amino acid sequence homologous oridentical to all or part of an antigen of the class I majorhistocompatibility complex (MHC-I) and, preferably, of the heavy chainof the latter.

[0025] All the cells of an organism have on their membrane moleculescalled histocompatibility antigens which define each individual. Thecorresponding genes, more than about ten, are located on chromosome 6 inhumans and exhibit high polymorphism, which makes it possible to ensurea high variability of these identity markers. There are two differentcategories of these histocompatibility antigens, classes I and IIrespectively, whose structure and functions are distinct. The class Imolecules, called HLA (for Human Leukocyte Antigen), are involved inpresenting antigenic peptides at the cell surface and play an essentialrole in the antiviral immune responses exerted by the cytotoxic Tlymphocytes.

[0026] The MHC-I molecules are heterodimers composed of a non-MHC lightchain designated β2-microglobulin (β2m) and a heavy chain encoded by theMHC genes, which are noncovalently linked. The heavy chain is a membraneprotein whose N-terminal part is oriented outside the cell whereas theC-terminal portion is cytoplasmic. The former comprises 3 domainsdesignated α1, α2 and α3 having about 90 amino acids in each. It isfollowed by a transmembrane region of about 25 amino acids and then theC-terminal region of about thirty amino acids. Most of the variationsbetween the products of the different alleles are located in the α1 andα2 domains, the α3 domain being relatively conserved-and β2m beinginvariable (for a review and the sequence comparison between the membersof the MHC-Is, see Bjorkman and Parham, 1990, Annu. Rev. Biochem. 59,253-288).

[0027] Among the polypeptides suitable for the purposes of the presentinvention, there may be mentioned more particularly the HLA A, B, C, D,E and F antigens or polypeptides derived therefrom.

[0028] In a particularly advantageous manner, the polypeptide in usewithin the framework of the present invention comprises a sequencehomologous or identical to all or part of the C-terminal region of theα2 domain of the MHC-I heavy chain and, more particularly, to the partcentered on the tryptophan residue at position 167, in particular thatextending from residues 156 to 180 (SEQ ID NO: 1). The numbering towhich reference is made is in accordance with that used, for example, inBjorkman and Parham (1990, supra).

[0029] According to another variant, a polypeptide in use within theframework of the present invention comprises an amino acid sequencehomologous or identical to at least 6 continuous amino acids of thesequence as shown:

[0030] in SEQ ID NO: 2 starting with the asparagine residue at position1 and ending with the asparagine residue at position 26,

[0031] in SEQ ID NO: 3 starting with the valine residue at position 1and ending with the asparagine residue at position 25,

[0032] in SEQ ID NO: 4 starting with the serine residue at position 1and ending with the arginine residue at position 25, and/or

[0033] in SEQ ID NO: 5 starting with the asparagine residue at position1 and ending with the serine residue at position 25.

[0034] A preferred polypeptide comprises an amino acid sequencehomologous or identical to fibronectin and, in particular, to at leastone of its type III modules and, in particular, to modules FNIII 1, 4, 5and/or 14. Of course, it may comprise several of them. It is alsopossible to envisage using human fibronectin or a peptide derivedtherefrom, for example, by mutation or fragmentation. As a guide, thefibronectin encoded by a single gene is a molecule which is involved inadhesion and cell contact phenomena. Its sequence and itscharacteristics are described in the literature accessible to personsskilled in the art (see in particular Bork and Doolittle, 1992, Proc.Natl. Acad. Sci. USA 89, 8990-8994 and Dickinson et al., 1994, 236,1079-1092). It is composed of 14 so-called type III modules (numberedfrom 1 to 14) whose primary sequence may vary, but whose β-sheetconformation is conserved.

[0035] According to a particularly advantageous embodiment, apolypeptide as defined above is more particularly intended to allow orto facilitate the attachment of a serotype C adenovirus to a host celland/or its entry into the latter. Among the adenoviruses which may beenvisaged, there may be mentioned more particularly serotypes 2 and 5.

[0036] The present invention also relates to a host cell capable ofexpressing a polypeptide in use within the framework of the presentinvention and its use to-allow or to facilitate the attachment of anadenovirus to its surface and/or the entry of the said adenovirus.Various types of host cells may be considered. They may be cells of anyorigin, for example of microorganisms, yeasts, insects, plants oranimals. A mammalian cell and, in particular, a human cell of theprimary or tumor type or derived from a line which can be cultured invitro will be preferred in particular. It may be of a hematopoietic(totipotent stem cell, leukocyte, lymphocyte, monocyte, macrophage andthe like), hepatic or renal origin, from the central nervous system,fibroblast, epithelial, pulmonary or muscular (myocyte, myoblast,satellite cell, cardiomyocyte and the like) origin. A particularlypreferred cell is or is derived from the 293 line established fromembryonic kidney cells by integration of the adenoviral E1 region(Graham et al., 1977, J. Gen. Virol. 36, 59-72). It is indicated thatthe expression of one or more polypeptides in use within the frameworkof the present invention at the surface of a host cell not usuallyexpressing the MHC-Is and/or fibronectin should allow its infectivity byan adenovirus. It could be used as a new cell producing adenoviralvectors. It is also possible to envisage the case of an overexpressionin a cell naturally expressing the said polypeptide. An overexpressingline derived from the 293 line should make it possible to improve theyields of production of an adenovirus of interest. Of course, thepolypeptide in use within the framework of the present invention may beassociated with the cell by chemical means or by means of a ligandrecognizing a cell surface protein. However, it is also possible toenvisage expression by recombinant DNA techniques. Such an embodiment iswithin the capability of persons skilled in the art. As a guide, thenucleotide sequence encoding the polypeptide in question may be isolated(by standard PCR or cloning techniques) or chemically synthesized beforebeing inserted into a conventional expression vector under the controlof appropriate regulatory elements, the vector being introduced into thehost cell by any prior art technique. The host cell in use within theframework of the present invention may also be modified so as tocomplement a defective adenovirus by transfection of (an) appropriatefragment(s) of adenoviral genome.

[0037] The subject of the present invention is also the use of a ligandcapable of influencing the attachment of an adenovirus to a host celland/or its entry into the latter, which are mediated by a polypeptide asdefined above. The ligand in use in the invention may be of any type.There may be mentioned, for example, the peptides, hormones, antibodiesor derivatives thereof and, in particular, single-chain antibodies ofthe scFv (for single chain fragment variable) type and soluble receptorslacking their transmembrane region. In particular, such a ligand may bederived from a polypeptide in use in the present invention. Inaccordance with the aims pursued by the present invention, the ligandmay have a negative (antagonist) or positive (agonist) influence.Preferably, a preferred ligand has a dissociation constant with respectto the adenovirus of between 0.01 and 100 nM, advantageously between 0.1and 50 nM, and most preferably between 0.5 and 10 nM.

[0038] In the case of an antagonist, the interaction of the ligand withthe fiber will make it possible to reduce or inhibit the process ofattachment and/or of entry of an adenovirus. In this context, aparticularly preferred ligand is based on a polypeptide as defined inSEQ ID NO: 1. By way of example, there may be mentioned a polypeptidecomprising an amino acid sequence homologous or identical to at least 6continuous amino acids contained in the sequence as shown in SEQ ID NO:6 starting with the arginine residue at position 1 and ending with thearginine residue at position 20. The use of the peptide designated MH20in the examples which follow will be preferred.

[0039] In the case of a positive influence, the ligand in use within theframework of the present invention is used to allow or to stimulate theattachment and/or the entry of adenoviruses. A ligand which is suitablefor the purposes of the invention comprises an amino acid sequencehomologous or identical to at least 6 continuous amino acids of thesequence as shown in SEQ ID NO: 7 starting with the arginine residue atposition 1 and ending with the serine residue at position 20. Apreferred example consists of the peptide designated below FN20.

[0040] The present invention also relates to a ligand comprising anamino acid sequence homogous or identical to at least 6 continuous aminoacids of the sequence as shown in SEQ ID NO: 6 or 7.

[0041] However, it may also be a ligand of adenoviral origin. Accordingto this embodiment, a preferred ligand is derived from the fiber of anadenovirus, in particular from the part of the head which interacts withthe abovementioned polypeptides. A peptide motif chosen in this regiontherefore ought to influence the infectivity of the adenoviruses withrespect to a host cell expressing the polypeptide. Advantageously, aligand covering residues 438 to 486 of the fiber of an adenovirus isused. More particularly, a ligand of a polypeptide as defined by SEQ IDNO: 1 is preferably derived from an Ad5 and comprises an amino acidsequence homologous or identical to at least 6 continuous amino acids ofthe sequence as shown in SEQ ID NO: 8, starting at the amino acidleucine at position 1 and ending at the amino acid aspartic acid atposition 18. A ligand which may also be envisaged may be derived fromthe fiber of a serotype 2 adenovirus and may comprise an amino acidsequence homologous or identical to at least 6 amino acids of thesequence as shown in SEQ ID NO: 9 starting at the threonine residue atposition 1 and ending at the valine residue at position 16.

[0042] The ligand of a polypeptide as defined by SEQ ID NO: 2 to 5 ismore particularly characterized by an amino acid sequence homologous oridentical to at least 6 amino acids of the sequence as shown in SEQ IDNO: 10 starting at the leucine residue at position 1 and ending at thethreonine residue at position 14 (Ad5) or in SEQ ID NO: 11 starting atthe asparagine residue at position 1 and ending at the asparagineresidue at position 13 (Ad2).

[0043] The subject of the present invention is also the use of a ligandaccording to the invention for the preparation of a medicament intendedto inhibit or reduce an infection by an adenovirus. In this context, theuse of an antagonist ligand with a therapeutic or prophylactic objectivewill be preferred. The use of a ligand according to the invention,preferably an agonist ligand, is appropriate for the preparation of amedicament intended to promote or facilitate an infection by anadenovirus, and in particular a recombinant adenovirus carrying atherapeutic gene intended for gene (curative) or anti-viral (AIDS) oranti-cancer therapy. Such a medicament finds it usefulness, for example,in association with gene therapy treatments so as to improve viralinfection in a patient treated with a recombinant adenovirus. It ispossible to envisage a parenteral or oral administration oralternatively administration by aerosol. The administration may be madein a single dose or a dose repeated once or several times after acertain period of time. The appropriate dosage and formulation varyaccording to different parameters, for example the individual, thedisease to be treated, the desired effect, the route of administrationor alternatively the adenovirus in question.

[0044] The present invention also relates to a method for selecting oridentifying a cellular receptor for a virus in an appropriate sample,comprising:

[0045] (a) the immobilization, onto an inert support, of a reagent ofviral origin comprising all or part of a surface protein of the saidvirus which determines its attachment to the cellular receptor,

[0046] (b) the incubation with the sample for a determined time,

[0047] (c) the elution of the sample retained in step (b) with all orpart of an antibody directed against the said reagent of viral origin,and

[0048] (d) the analysis of the sample eluted in step (c).

[0049] The inert support may be, without limitation, in any form (cone,tube, well, beads and the like) and may be made of any material(natural, synthetic, such as polymers, chemically modified or otherwiseand the like). The attachment of the reagent to the inert support may becarried out in a direct or indirect manner. In a direct manner, theprocedure will be preferably carried out by adsorption, that is to saynoncovalently, although the establishment of covalent bonds may also beconsidered. In an indirect manner, an anti-reagent compound capable ofinteracting with the reagent may be attached beforehand so as toimmobilize the whole onto the inert support. According to anadvantageous embodiment, the sample consists of a so-called randomlibrary, and in particular an expression library (genomic fragments,cDNA) or a peptide library or, preferably, a phage library expressingpeptide motifs (phagotopes). Such libraries are described in theliterature or are commercially accessible. With the aim of selecting oridentifying a cellular receptor for an adenovirus, there is preferablyused as reagent of viral origin all or part of the fiber and, inparticular, of the head of an adenovirus and, as eluent, an anti-fiberneutralizing antibody (inhibitor of the attachment of the virus to thesurface of the host cell). The fiber or its fragments may be produced bythe recombinant route and the antibodies by the hybridoma technique orby genetic engineering (production of single chain antibody scFv, Faband the like). It is indicated that most anti-fiber antibodies areneutralizing. The analysis is carried out by comparing the sequence ofthe eluted sample with data banks. Such an analysis is within thecapability of persons skilled in the art.

[0050] Finally, the present invention also relates to a method forselecting or identifying the part of a viral protein which determinesthe attachment of a virus to a cellular receptor in an appropriatesample, comprising:

[0051] (a) the immobilization, onto an inert support, of all or part ofan antibody directed against the said viral protein,

[0052] (b) the incubation with the said sample for a determined time,

[0053] (c) the elution of the sample retained in step (b) with a reagentof viral origin comprising all or part of the said viral protein, and

[0054] (d) the analysis of the sample eluted in step (c).

[0055] The specific embodiments cited above may also apply in thiscontext.

[0056] The subject of the present invention is also the use of abifunctional ligand for targeting an adenovirus to a cell surfaceprotein other than the natural cellular receptor for the saidadenovirus, the said bifunctional ligand comprising a first ligand partcapable of interacting with the fiber of the said adenovirus, a secondligand part capable of interacting with the said cell surface proteinand, optionally, a spacer between the said first and second ligandparts.

[0057] For the purposes of the present invention, a bifunctional ligandis capable of interacting with two different species, one preferablysituated at the surface of an adenovirus and the other at the surface ofa host cell, at the level of a cell surface protein other than thenatural cellular receptor for the said adenovirus. Moreover, the twoligand parts may be optionally separated by a spacer comprising from oneto about fifteen amino acids which are preferably not charged. Ofcourse, the order of the species is of no importance, it being possiblefor the domain interacting with the adenoviral protein to be at the N orat the C terminus of the bifunctional ligand, the C-terminal positionbeing preferred. The use of such a bifunctional ligand makes it possibleto target an adenovirus to a host cell of interest, for example a tumorcell, an infected cell, a particular cell type or a category of cellscarrying a specific surface marker. After binding of the saidbifunctional ligand to the cell surface protein, the species recognizingthe adenoviral protein is exposed, which should create “lures” of viralreceptors of the same type as the primary receptors (α2 domain of theMHC-Is) and create or increase the number of adenovirus primaryreceptors at the surface of the host cell.

[0058] Preferably, the ligand part interacting with the adenoviral fiberhas the characteristics of the ligand defined above; it is in particularderived from the α2 domain of the MHC-I and preferably comprises anamino acid sequence homologous or identical to at least 6 continuousamino acids contained in SEQ ID NO: 6. Still more preferably, itconsists of the peptide MH20 (SEQ ID NO: 6).

[0059] As regards the ligand part interacting with the cell surfaceprotein, it is adapted to the host cell which it is desired to target.In the case of a cell infected by the HIV virus (Human ImmunodeficiencyVirus), the ligand may be an antibody fragment against fusin, the CD4receptor or against an exposed viral protein (envelope glycoprotein) oralternatively the part of the TAT protein of the HIV virus extendingfrom residues 37 to 72; (Fawell et al., 1994, Proc. Natl., Acad. Sci.USA 91, 664-668). In the case of a tumor cell, the choice will be for aligand recognizing a tumor-specific antigen (MUC-1 in the case of breastcancer, antigens of the papillomavirus HPV and the like) or one which isoverexpressed (IL-2 receptor overexpressed in certain lymphoid tumors).If it is desired to target the T lymphocytes, it is possible to use a Tcell receptor ligand. Moreover, transferrin is a good candidate forhepatic targeting. There may also be mentioned the peptide EGF(abbreviation for Epidermal Growth Factor) which allows targeting tocells expressing the EGF receptor or the GRP peptide (for GastrinReleasing Peptide) having the sequence GNHWAVGHLM (Michael et al., 1995,Gene Ther. 2, 660-668) which binds to the GRP cellular receptor. Ingeneral, the ligands which may be used in the context of the inventionare widely described in the literature.

[0060] A bifunctional ligand in use within the framework of the presentinvention may be obtained by recombinant DNA techniques, by synthesis orby chemical coupling of the two parts of the ligands in question.Preferably, the adenovirus to be targeted is recombinant and carries acytotoxic gene or is capable of inducing cellular apoptosis. Such genesare perfectly known. There may be mentioned, in particular, the geneencoding thymidine kinase of the HSV-1 virus (herpes simplex virus type1).

[0061] By way of preferred examples, there may be mentioned abifunctional ligand comprising the peptide MH20 and GRP. The MH20 andGRP peptide domains may be inversely oriented: respectively MH20-GRPwhen MH20 is at the N-terminal position and GRP-MH20 when MH20 is at theC-terminal position. Another embodiment uses a ligand having an MH20species and an antibody species of the ScFv (Single Chain FV fragment)type. According to a particularly preferred embodiment, the saidbifunctional ligand comprises an amino acid sequence homologous oridentical to all or part of the sequence as shown:

[0062] (i) in SEQ ID NO: 22 starting at the arginine residue at position1 and ending with the methionine residue at position 35, or

[0063] (ii) (ii) in SEQ ID NO: 23 starting at the lysine residue atposition 1 and ending with the arginine residue at position 35.

[0064] The present invention also relates to a bifunctional ligand asdefined above and, in particular the ligands GRP-MH20 or MH20-GRP. Theligand GRP-MH20 is particularly preferred.

[0065] The subject of the present invention is also a cell carrying atits surface such a bifunctional ligand. The advantage of such a cell isto increase the number of primary receptors of the MHCI-α2 type. Thesaid cell is advantageously a mammalian cell of any origin (see above).It is preferably a cell for complementation of an adenovirus defectivefor the E1 function and optionally for another function (E2, E4, E2 andE4, and the like, see Application WO94/28152). A preferred example isthe 293 line. It may be generated by recombinant DNA techniques(expression of the bifunctional ligand by means of an appropriate vectorcomprising the elements allowing expression at the cell surface, forexample signal sequence and/or transmembrane region), by covalent ornoncovalent chemical bonding or by simple interaction between the celland the ligand.

[0066] The present invention is illustrated with reference to thefollowing figures.

[0067]FIG. 1 presents the phagotopes obtained after biopanning using theantibody 1D6.3 (a) or 7A2.7 (b) as ligand. The peptide motifs of thephagotopes are aligned with respect to the sequence of the Ad5 head (theinitiator methionine residue of the fiber representing +1. The regionsforming the β sheet structures (Xia et al., 1994, supra) are underlinedand indicated by (D), (E) and (F). The residues which are identical orconserved in the sequences are indicated in bold.

[0068]FIG. 2 presents the phagotopes obtained after biopanning using (a)the antibody 7A2.7 or (c) 1D6.3 as eluent, (b) and (d) the consensussequences determined from the phagotopes (a) and (c) respectively aswell as the homologous sequences found by analyzing the SWISS PROT databank. The residues conserved at analogous positions are indicated inbold.

[0069]FIG. 3 illustrates the expression of the luciferase gene in HeLacells infected with the Ad5Luc3 virus at a constant MOI (0.16 pfu/10⁵cells). ( (∘) the Ad5Luc3 is added to the cells cooled to 0° C. in thepresence of increasing molarities of peptide (a) FN20 (0 to 500 μM or(b) MH20 (0 to 50 μM). The controls correspond to the incubation of thepeptides after the attachment of Ad5Luc3 (Δ) or after endocytosis (□).

[0070]FIG. 4 illustrates the expression of the luciferase gene inDaudi-HLA− () or Daudi-HLA+ (∘) cells infected with increasingconcentrations of Ad5Luc3 (0.3 to 150 pfu/10⁵ cells). The Ad5Luc3 isbrought into contact with the cells precooled to 0° C. for 1 h in orderto allow viral attachment but not the entry. The luciferase activity isevaluated after 18 h of culture at 37° C. The RLU values represent themean of three separate experiments.

[0071]FIG. 5 illustrates the principle of the bifunctional ligand methodmimicking the primary receptors for the adenovirus.

[0072]FIG. 6 represents the luciferase enzymatic activity as a functionof the MOI of Ad5Luc3 on NIH cells (a), Swiss 3T3 cells (b) and HeLacells (c).

EXAMPLES

[0073] The HeLa cells (ATCC CCL2) are cultured in monolayers accordingto the prior art techniques. A DMEM medium (Dulbecco's Modified Eagle'sMedium; Gibco) containing 10% heat-inactivated fetal calf serum (FCS),L-glutamine and the usual antibiotics, is preferably used. The DaudiHLA− (ATCC CCL213) and HLA+ (Quillet et al., 1988, J. Immunol. 141,17-20) cells are maintained in RPMI 1640 medium (Gibco) supplementedwith 15% FCS.

[0074] The wild-type Ad5 and the recombinant Ad5Luc3 are propagatedbetween the HeLa cells by the standard techniques. As a guide, Ad5Luc3is a replication-competent adenovirus which contains the luciferase geneplaced under the control of the SV40 virus (simian virus 40) earlypromoter inserted into the E3 region of the adenoviral genome (Mittal etal., 1993, Virus Research 28, 67-90).

Example 1 Production of Monoclonal Antibodies (mAb) which is Capable ofInhibiting the Attachment of Ad5 to the Permissive Cells

[0075] The murine mAbs 1D6.3 and 7A2.7 were generated by conventionaltechniques by injecting the head of the Ad5 fiber produced in bacteriaby the recombinant route (Henry et al., 1994, J. Virol. 68, 5239-5246;Douglas et al., 1996, Nature Biotech 14, 1574-1578) into Balb/C mice.The fusion and the production of hybridoma clones are conventionaltechniques within the capability of persons skilled in the art. Thesecreting clones are selected by their recognition of the antigen whichserved for the immunization in ELISA. It is indicated that they exhibita neutralizing activity toward the virions (Michael et al., inpreparation).

[0076] Sero-Reactivity of the Monoclonal Antibodies 1D6.3 and 7A2.7

[0077] The reactivity of the antibodies is tested with respect to thedomain of the head of the fiber of 3 different serotypes (Ad2, Ad5 andAd3) which is prepared by the recombinant route. The correspondingsequences are isolated by PCR (Polymerase Chain Reaction) from viralgenomic DNA and then introduced into the AcNPV virus (Autographacalifornica Nuclear Polyhedrosis Virus) under the control of thepolyhedrin promoter (Luckow and Summer, 1989, Virology 170, 31-39). Therecombinant proteins are expressd in the Sf9 (Spodoptera frugiperda)insect cells. The general technology is detailed in Karayan et al.(1994, Virology 202, 782-796) and Novelli and Boulanger (1991, Virology185, 365-376). More precisely, the Ad5 sequences carrying the lastrepeated motif of the stem followed by the head of the fiber are clonedwith the aid of the primers represented in SEQ ID NO: 12 and 13. Thesense primer corresponds to nucleotides 32164 to 32205 of the Ad5 genome(Chroboczek and Jacrot, 1987, Virology 161, 549-554), includes 4mismatches so as to create a BamHI site and to replace the threonine atposition 388 of the native fiber with an initiator ATG codon. Theantisense primer corresponds to nucleotides 32919 to 32883 of the Ad5genome and makes it possible to create a KpnI site in order tofacilitate subsequent cloning steps. The recombinant protein harvestedin the Sf9 cell supernatants is designated F5-AT386. The Ad2 head isproduced from the baculovirus vector described in Louis et al. (1994, J.Virol. 68, 4104-4106). The expression product designated F2-AT388 startsat position 388 (by replacement of the Ala of the native sequence with aMet) and carries, in addition to the head domain, the last repeatedmotif of the stem. Finally, for the corresponding sequences of Ad3, asense primer (SEQ ID NO: 14) is used which is designed to introduce anNcoI cloning site and to replace the Asn and Ser codons at position 124and 125 with the Met and Ala codons respectively. The antisense primer(SEQ ID NO: 15) introduces a KpnI site. The expression product isdesignated F3-AT124.

[0078] The wells of an ELISA plate are coated with the recombinantprotein F5-AT386, F2-AT388 or F3-AT124 with which the mAb 1D6.3 or 7A2.7and then a labeled anti-mouse antibody (for example with phosphatase orperoxidase) are reacted. A positive reaction is observed toward thenative recombinant protein F5-AT386. No reaction is detected in thewells containing the protein F5-AT386 denatured with SDS or in thosecontaining the native or denatured products F2-AT388 and F3-AT124. Thesedata suggest that these antibodies recognize a serotype C-specificconformational epitope.

[0079] Inhibitor Effect of the Monoclonal Antibodies 1d6.3 and 7A2.7 onthe Cellular Attachment of Ad5.

[0080] A test for microbinding onto HeLa cells in culture is carried outwith the aid of Ad5 virions labeled with [¹⁴C]valine (specific activityof 2200 to 2500 cpm/10⁸ virions) followed by autoradiography in situ(Silver and Anderson, 1988, Virology 165, 377-387). To do this, thecells at the semiconfluent state are placed in the presence of aconstant quantity of radioactive virions (10³ cpm per 5×10⁴ cells) at amultiplicity of infection (MOI) of 1000 virions per cell for 1 h at 0°C. in the presence of mAb 1D6.3 or 7A2.7 (1:10, 1:8, 1:4 and 1:2dilutions of the respective hybridoma supernatants whose mAbconcentration is estimated at 0.1-0.2 μg/ml, which corresponds to anexcess of mAb relative to the virions present in the inoculum of 100,250, 500 and 1000 respectively). The cells are then washed in thepresence of PBS, fixed with 0.1% paraformaldehyde in PBS, dried andcovered with the K4 emulsion in gel form (Ilford Nuclear Research).After an exposure of one week and developing (developing agent D19B,Kodak), the samples are briefly stained with 0.5% toluidine blue, takenup in H₂O and examined under a microscope. The density of the reducedsilver crystals around the contour of the cells is representative of thenumber of [¹⁴C] virions bound to the cell surface.

[0081] In the absence of an anti-head mAb or at a low concentration(1:10 dilution), a dark halo of reduced silver crystals is visiblearound the cells, indicating adsorption of the virions to their surface.A reduction in the halo which is dependent on the mAb concentration isobserved for the 1:8, 1:4 and 1:2 dilutions. These results reflect ablocking of the binding of the adenoviral head to the primary cellularreceptor due to the 1D6.3 and 7A2.7 mAbs directed against this part ofthe fiber. Comparison of the surface area of the halos for the samedilutions shows that the 1D6.3 antibody is more inhibitory of theattachment of Ad5 to the HeLa cellular receptor than the mAb 7A2.7.

Example 2 Identification of the Epitopes for the mAb 1D6.3 and 7A2.7

[0082] The epitopes of the fiber being assumed to be conformational(Fender et al., 1995, Virology 214, 110-117), the conventional method ofidentification of epitopes by scanning of peptides is not appropriate.The procedure is carried out according to a biopanning technique derivedfrom those described by Smith and Scott (1993, Methods Enzymol. 217,228-257) and Hong and Boulanger (1995, EMBO J. 14, 4714-4727). In thiscase, the mAb 1D6.3 or 7A2.7 is adsorbed overnight at 4° C. onto amicrotiter plate (Nunc Immunomodule MaxiSorp F8) at a concentration of 1μg/well in a 0.1 M sodium carbonate buffer pH 9.6. The immobilizedantibodies are brought into contact with a phage library expressinghexapeptides (fUSE5 phages; Scott and Smith, 1990, Science 249,386-390). In a second step, the phages retained are eluted either with aconventional acidic elution buffer or, more selectively, by competitionin the presence of an excess of recombinant protein F5-AT386. Thehexapeptide motifs (phagotopes) carried by the eluted phages aredetermined by sequencing the protein pIII fUSE5 by the method of Sangeret al. (1977, Proc. Natl. Acad. Sci. USA 74, 5463-5467). The sequencehomologies with the phagotopes are searched out in the Swiss Prot databank and the FASTA 1.6 program (Pearson and Lipman, 1988, Proc. Natl.Acad. Sci. USA 85, 2444-2448) and the sequence alignments made usingversion W(1;4) of the Clustal program (Higgins and Sharp, 1988, Gene 73,237-244).

[0083] As shown in FIG. 1, the mAb 1D6.3 retains different phagotopeswhose sequences overlap, and are homologous to the Ad5 head extendingbetween the Val residue at position 438 and the Asp residue at position462. In spite of a degree of degeneration and dispersion, it waspossible to determine a central motif of sequence LAPISGTVQSAHLIIRFDcorresponding to the amino acids at positions 445 to 462 (SEQ ID NO: 8).This motif is centered on the His residue at position 456, whichcorroborates the presence of histidine in several independentphagotopes. Furthermore, three phagotopes contain a hisitidine close toa serine (GISHTG and GASHTV) and a homologous sequence is found in thehead (⁴⁵³QSAHLI⁴⁵⁸). On the N-terminal side, the sequence LAPIS isrepresented in several phagotopes in the form L-P, VAP-S and LIPFNS.

[0084] The sequence analyses of the 15 phagotopes retained by the mAb7A2.7 demonstrated the presence of a proline on 4 occasions, oftryptophan and histidine 8 times and the association in the samephagotope of two aromatic residues is found five times. It thereforeseems that the epitope for the mAb 7A2.7 contains a proline residue,such as the one at position 475 of the fiber, near a group of aromaticresidues, such as ⁴⁷⁷YWNF⁴⁸⁰. In addition, two phagotopes FWLAVR andWALFRS are homologous to the motif ⁴⁷⁷YWNFR⁴⁸¹. On the basis of thesedata, the epitope for the mAb 7A2.7 was mapped between residues 473 and486 of the Ad5 fiber (SEQ ID NO: 10).

[0085] In short, the mAbs 1D6.3 and 7A2.7 recognize adjacent segments of15 to 20 aa in the linear sequence of the Ad5 head, the residuesextending from positions 445 to 462 and 473 to 486 respectively.According to the three-dimensional model of the head proposed by Xia etal. (1994, Curr. Biol. Structure 2, 1259-1270), the two epitopes occupyregions which are continuous from a spatial point of view. The epitopefor the mAb 1D6.3 covers part of the CD loop and of the β sheet D,whereas the epitope for the mAb 7A2.7 is located at the level of theadjacent segment DE and of the two β sheets E and F. The 1D6.3 epitopeis situated inside a sheet R whereas the 7A2.7 epitope is oriented moresideways relative to the sheet R.

Example 3 Identification of the Cellular Receptors for Ad5 by theReverse Biopanning Technique

[0086] This technique is termed reverse relative to the preceding onesince the fiber is used as ligand and the mAb as eluent. The procedureis therefore carried out by immobilizing the head of the Ad5 fiber withwhich the phage library expressing hexapeptide phagotopes is reacted.The phages adsorbed may be eluted either with a conventional acidicbuffer or with the mAb 1D6.3 or 7A2.7 and the recombinant protein pIIIcarrying their respective phagotopes is sequenced. The search in databanks is carried out as above. The hexapeptide motifs identified arepresented in FIG. 2.

[0087] The phagotopes produced by competition with the mAb 7A2.7 arepresented in FIG. 2a. Their analysis makes it possible to derive aconsensus sequence (FIG. 2b) which exhibits homology with the motifs 1,3, 5 and 14 of the type III module of human fibronectin (SEQ ID NO: 2 to5) (Main et al., 1992, Cell 71, 671-678). They are situated at the levelof the β sheet B and of the adjacent BC loop of the FNIII module(Dickinson et al., 1994, J. Mol. Biol. 236, 1079-1092).

[0088] The phagotopes eluted after the action of the mAb 1D6.3 aredescribed in FIG. 2c. All the sequences overlap and make it possible toalso determine a consensus sequence (FIG. 2d). The search for homologywith the sequences listed in data banks reveals homology with theC-terminal region of the α-2 domain of the heavy chain of the class IMHC molecules (MHC-1α-2) (position 156 to 180) (SEQ ID NO: 1).

Example 4 Interactions of the Adenoviral Fiber with the FNIII Model andthe MHC-1α-2 Domain

[0089] The interaction is studied in vitro with the aid of a chimericprotein derived from the C-terminal fusion of the GST (GlutathioneS-transferase) protein to a pentadecapeptide RHILWTPANTPAMGY reproducingthe consensus sequence homologous to the β sheet B and the BC loop ofFNIII (see preceding example and FIG. 2b). To do this, theoligonucleotides presented in the sequence identifiers 16 and 17 arehybridized and introduced into the XhoI site of the plasmid pGEX-KG(Guan and Dixon, 1991, Anal. Biochem. 192, 262-267). It should be notedthat the oligonucleotides, once rehybridized, generate an XhoI site atthe 5′ end if the insert is cloned in the correct orientation. Thismakes it possible to integrate a single copy or multiple copies intandem at the level of the reconstituted XhoI site. The sequence of thefusion product comprising a copy of the pentadecapeptide (designatedGST-FNx1) may be schematically represented in the following manner: GST-(site for cleavage bythrombin-PGIS-GGGGG-ILDSMGRLE-RHILWTPANTPAMGY(V)-ELKLNS-stop. Theconstructs GST-FNx2 and GST-FNx3 comprise respectively 2 and 3 repeatsof the pentadecapeptide between residues LE and EL of the cloningcassette.

[0090] The sense and antisense oligonucleotides (SEQ ID NO: 18 and 19)encoding the consensus sequence obtained by competition with the mAb1D6.3 (FIG. 2d) are inserted according to the same strategy as above atthe C-terminal end of GST to give the constructs GST-MHCx1, GST-MHCx2and GST-MHCx3 depending on the number of motifs present. The chimericproteins GST-FN and GST-MHC are produced in E. coli, extracted andaffinity purified on agarose-glutathione beads (Sigma) according toconventional methods (Smith and Johnson, 1988, Gene 67, 31-40).

[0091] In parallel, the complete fibers of serotypes 2, 3 and 5 areproduced by the recombinant route according to the baculovirus/Sf9 celltechnology already used. The construct F2-FL582 carrying the gene forthe Ad2 fiber is described in Novelli and Boulanger (1991, Virology 185,365-376). The sequences encoding the Ad5 and Ad3 fibers are isolated byPCR using the viral DNA as template and appropriate sense and antisenseprimers such as those listed in SEQ ID NO: 20 and 13 and 21 and 15. Theamplified segment is introduced into a baculovirus vector under thecontrol of the polyhedrin promoter and the expression product recoveredin the culture supernatants. F5-FL581 and F3-FL320 corresponding to theAd5 and Ad3 fibers respectively are obtained.

[0092] The capacity of the FNIII module and of the recombinant α-2 MHC-1domain to bind the adenoviral fiber is evaluated by immunotransfer afterin vitro incubation of the fusion proteins GST-FN and GST-MHC and of therecombinant fibers F2-FL582, F5-FL581 and F3-FL320. The complexes formedare isolated on agarose glutathione beads under the conditions describedby Johnson et al. (1995, J. Biol. Chem. 270, 24352-24360) and thenanalyzed on 12.5% polyacrylamide gel (SDS-PAGE) using a discontinuousbuffer system (Laemmli, 1970, Nature 227, 680-685). The proteins aretransferred onto a nitrocellulose membrane (0.8 mA/cm²; CambridgeElectrophoresis system, UK) in a 25 mM Tris-HCl, 192 mM glycine buffer,pH 8.3 containing 20% methanol. After treating with a blocking solution(5% skimmed milk, 1 calf serum in TBS-T buffer: 20 mM Tris-HCl pH 7.8,0.15 M NaCl, 0.05% Tween 20), the membrane is brought into contact withthe antibody 4D2.5 (Hong and Engler, 1991, Virology 185, 758-767) andthen a horseradish peroxidase-labeled anti-IgG conjugate. The revealingby the chemiluminescent peroxidase substrate (SuperSignal, PierceChemicals) is carried out according to Carrière et al. (1995, J. Virol.69, 2366-2377) and the luminograms (Hyperfilm β-max, Amersham) areanalyzed at 610 nm with the aid of an automated densitometer (REP-EDC,Helena Laboratories, Beaumont, Tex.). As a guide, the antibody 4D2.5recognizes the FNPVYP epitope of the tail domain conserved in mostmammalian adenoviruses.

[0093] GST-FNx1, GST-FNx2 and GST-FNx3 bind to F5-AT386 and F2-AT388with high efficiency whereas F3-AT124 is retained to a lesser extent. Asimilar behavior is observed with the chimeric proteins GST-MHCx1,GST-MHCx2 and GST-MHCx3 which retain F5-AT386 and F2-AT388 with a higherefficiency than F3-AT124.

[0094] The Ad5 fiber binds to the fusion proteins GST-FN and GST-MHCwith an affinity two to three times greater compared with the Ad2 fiberand ten to fifteen times greater compared with the Ad3 fiber.Furthermore, the binding efficiency is not dependent on the number ofmotifs present in the fusion protein, the intensity being comparable andeven sometimes lower between GST-FNx1 and GST-FNx3 and GST-MHCx1 andGST-MHCx3. This may be explained by the fact that the motifs in tandemmay adopt a conformation which interferes with the binding.

Example 5 Influence of the Synthetic Peptides Derived from FNIII andMHC-1α-2 on the Attachment of Viruses to the Cell Surface

[0095] Two synthetic peptides reproducing the FNIII and MHC-Iα2 motifswere chemically synthesized and purified according to the prior arttechniques. FN20 (SEQ ID NO: 7) reproduces the consensus sequence of thephagotopes eluted by the antibody 7A2.7 and MH20 (SEQ ID NO: 6)corresponds to that of the phagotopes eluted by the mAb 1D6.3.

[0096] The peptides FN20 and MH20 are tested with respect to theattachment of the reporter adenovirus Ad5Luc3 to HeLa cells cultured invitro. The test is carried out in part at 0° C., a temperature whichallows the attachment of the viruses to the surface of the permissivecells but, on the other hand, blocks the entry of the viruses and therecycling of the receptors. Ad5Luc3 (MOI 0.16 pfu/10⁵ cells) ispreviously incubated with increasing quantities of peptides (0.01 to 500nM) at room temperature for 2 hours and the mixture is then added to acell culture placed on ice. After 1 h at 0° C., the nonadsorbed virusesand the peptides are removed by washing and the culture is continued for18 h at 37° C. after adding a preheated medium. The cellular lysates areprepared in a conventional manner and the luciferase activity expressedin RLU (for Relative Light Units) is determined (Promega substrate,Madison, Wis.; Lumat LB-9501 luminometer, Brethold Bioanalytical,Wildbad, Germany).

[0097] The results of competition with the peptide FN20 are presented inFIG. 3a. No significant effect is obtained up to a molarity of 10 μM andthen a gradual increase in the luciferase activity appears above 25 μM.In particular, the activity increases by a factor of 100 between 25 and100 μM. The peptide FN20 therefore has a stimulatory effect on the viralattachment. It confers no apparent cytotoxicity and has no negativeeffect on the expression of the luciferase gene once the virus ispreattached (peptide added to the cell culture after the step of viralattachment at 0° C.) or preendocytosed (peptide added to the cellculture after the step of attachment and of penetration of the virus).

[0098] In the case of the peptide MH20 (FIG. 3b), a slight increase inthe expression of the luciferase gene is observed for molarities ofbetween 0.05 and 2.5 μM (activity five to six times greater at 2.5 μM).This phenomenon is followed by a rapid decrease in the luciferase levelswhen the molarities used are greater than 5 μM, with a decrease by afactor of 100 compared with the control at 25 μM and by almost fourorders of magnitude at 50 μM, showing that bound to the virus, it blocksthe attachment to the cell receptor. As above, the peptide MH20 atconcentrations of 50 μM, exhibits no cytotoxic effect and does notinfluence the expression of the reporter gene after preattachment orpreendocytosis of Ad5Luc3. The practically complete inhibition of theluciferase activity in the presence of 50 μM of MH20 is a reflection ofcomplete neutralization of the virus.

Example 6 Sero-Specificity of the Viral Neutralization by the SyntheticPeptides

[0099] The wild-type adenoviruses Ad5, Ad2 Ad3 are preincubated for 2 hat room temperature with the inhibitory peptide MH20 at a constantmolarity (25 μM), the MOI varying from 0.2 to 2 pfu/cell. The mixture isplaced in the presence of the HeLa cells for 1 h at 0° C. and theculture continued at 37° C. after removal of the nonadsorbed virusesunder the same conditions as those described above. The level ofsynthesis of the hexon, of the 100 k protein, of the penton base is ofthe fiber is estimated by immunotitration (Wohlfart, 1988, J. Virol. 62,2321-2328) on cellular extracts collected 48 h after infection.

[0100] The infection of the HeLa cells with Ad5 or Ad2 at an MOI of 0.2to 2 pfu/cell in the presence of 25 μM of MH20 during the virusattachment phase is followed by inhibition of the synthesis of theproteins of the viral capsid hexon, look protein, penton base and fiber48 h after the infection (factor 15 to 30). On the other hand, when theinfection is carried out under the same conditions with the wild-typeAd3, the synthesis of the structural proteins is only reduced by afactor of 1.5 to 2, which suggests that the adenoviral neutralization byMH20 is serotype-dependent.

Example 7 Affinity of the Ad5 Fiber for the Peptides FN20 and MH20

[0101] 5, 10 and 25 μM of synthetic peptides FN20 or MH20 areimmobilized on the polystyrene surface of a 96-well microtiter plate(Nunc, Maxisorb) overnight at 4° C. After washing and then blocking witha 3% solution of bovine serum albumin (BSA) in PBS buffer, increasingconcentrations of F5-FL581 fibers taken up in PBS buffer andradioactively labeled (labeling with [³⁵S]methionine and [³⁵S]cysteine;specific activity of 50,000 to 65,000 cpm/μg of protein) are caused toreact. The fiber adsorbed onto either of the peptides is eluted with asuitable solution (1 M urea, 1 M NaOH and 1% SDS) and then precipitatedin the presence of trichloroacetic acid. The radioactivity content inthe precipitate recovered on the GF/C filter is counted with the aid ofa liquid scintillation spectrometer (Beckman LS-6500) and thedissociation constants (Kd) determined according to Scatchard (1949,Annls NY Acad. Sci. 51, 660-672).

[0102] The Kd of the labeled Ad5 fiber with respect to the peptide MH20is evaluated at 3.0±0.6 nM and that found for the peptide FN20 is8.0±1.9 nM (n=3 in both cases).

Example 8 The Infectivity of Ad5 is Dependent on the Expression of theMHC-I at the Surface of the Permissive Cells

[0103] The Daudi line of B lymphoblastoids, which is established from aBurkitt's lymphoma, is naturally deficient in the expression ofβ₂-microglobulin and, as a result, does not possess at its surface classI HLA molecules (Daudi HLA−). The cell line E8.1 derived from the Daudiwas generated by transfection of a gene encoding α₂-microglobulin so asto restore the expression of the class I HLA molecules at their surface(Daudi-HLA+; Quillet et al., 1988, J. Immunol. 141, 17-20).

[0104] The experiments for attachment of Ad5Luc3 at 0° C. were carriedout on Daudi HLA− and Daudi-HLA+ cells with an MOI of three orders ofmagnitude greater than that used in the case of HeLa cells (0.3 to 150pfu/10⁵ cells). The luciferase activity measured in the cellular lysates18 h post-infection is represented in FIG. 4. When the infection relatesto Daudi-HLA+ cells, the luciferase activity increases regularly in anMOI-dependent manner until a plateau is reached above 5 pfu/10⁵ cells.As regards the Daudi HLA− cells, the luminescent signal is very low (3to 4 orders of magnitude) compared to that observed with the cellsprovided with functional HLA molecules at their surface. Theseexperiments show that the expression of MHC-I at the cell surface isnecessary for the Ad5 infection.

Example 9 Bifunctional Ligand Mimicking the Primary Receptors for theAdenovirus

[0105] This example describes the construction of a bifunctional peptidewhich contains two domains, one recognizing the head of the adenoviralfiber and the other a cell surface protein. A peptide is used below inwhich MH20 is fused to GRP (Gastrin Releasing Peptide). Twoconstructions are possible, orienting the two peptide domains in areversed manner, N- versus C-terminus, giving rise to the peptideMH20-GRP (SEQ ID No: 22) and to the peptide with the reverse orientationGRP-MH20 (SEQ ID No: 23).

[0106] As illustrated in FIG. 5, the binding of this peptide to the cellsurface GRP receptors should create “lures” of viral receptors of thesame type as the primary receptors, that is to say the α2 domain of theMHC class I molecules. The apparent result is to create or to increasethe number of primary viral receptors for the adenovirus at the surfaceof cells possessing GRP receptors.

[0107] The human cells (HeLa) or murine cells (Swiss-3T3 or NIH-3T3,ATCC CRL-1658) are rinsed and incubated between 0 and 4° C. with anisotonic solution (PBS) containing 500 μM of peptide. After 1 h, thesolution is removed and replaced with the viral inoculum (Ad5Luc3)according to a protocol already described (see above or Hong et al.,1997, EMBO J. 16, 2294-2306). The virus is incubated for 1 hour between0 and 4° C. to allow its attachment, and then the excess nonadsorbedvirus is rinsed with culture medium precooled to 4° C. and the cells areagain placed at 37° C. in the presence of culture medium preheated to37° C. The virus is endocytosed at 37° C. and the viral cycle then lastsfor 18 to 20 h at 37° C.

[0108] As shown in FIG. 6, it is observed that the curves obtained inthe absence of any peptide (control curves indicated: -peptide) andthose in the presence of peptide in the MH20 orientation N-terminusside-bound to GRP at the C-terminus (MH20-GRP) have an identical slope.

[0109] On the other hand, in the case of the reverse orientation, GRP onthe N-terminus side, and MH20 on the C-terminus side, the increase inthe number of adsorbed viruses is significant, as shown by the increasein the luciferase activity: eight to ten times for NIH-3T3, five to sixtimes for the HeLa cells and two to three times for the Swiss-3T3. Thebinding of the GRP portion of the bifunctional peptide, ligand for theGRP receptors, made it possible to increase the apparent number ofreceptors for the Ad5 fiber of the alpha 2 domain type of the MHC classI molecules (MHC I-α2).

1 98 1 25 PRT Homo sapiens 1 Leu Arg Ala Tyr Leu Glu Gly Thr Cys Val GluTrp Leu Arg Arg Tyr 1 5 10 15 Leu Glu Asn Gly Lys Glu Thr Leu Gln 20 252 26 PRT Homo sapiens 2 Asn Ser His Pro Ile Gln Trp Asn Ala Pro Gln ProSer His Ile Ser 1 5 10 15 Lys Tyr Ile Leu Arg Trp Arg Pro Lys Asn 20 253 25 PRT Homo sapiens 3 Val Lys Val Thr Ile Met Trp Thr Pro Pro Glu SerAla Val Thr Gly 1 5 10 15 Tyr Arg Val Asp Val Ile Pro Val Asn 20 25 4 25PRT Homo sapiens 4 Ser Thr Val Leu Val Arg Trp Thr Pro Pro Arg Ala GlnIle Thr Gly 1 5 10 15 Tyr Arg Leu Thr Val Gly Leu Thr Arg 20 25 5 25 PRTHomo sapiens 5 Asn Ser Leu Leu Val Ser Trp Gln Pro Pro Arg Ala Arg IleThr Gly 1 5 10 15 Tyr Ile Ile Lys Tyr Glu Lys Pro Ser 20 25 6 20 PRTArtificial Sequence Synthetic peptide MH20 6 Arg Ala Ile Val Gly Phe ArgVal Gln Trp Leu Arg Arg Tyr Phe Val 1 5 10 15 Asn Gly Ser Arg 20 7 20PRT Artificial Sequence Synthetic peptide FN20 7 Arg His Ile Leu Trp ThrPro Ala Asn Thr Pro Ala Met Gly Tyr Leu 1 5 10 15 Ala Arg Val Ser 20 818 PRT Mastadenovirus, serotype 5 8 Leu Ala Pro Ile Ser Gly Thr Val GlnSer Ala His Leu Ile Ile Arg 1 5 10 15 Phe Asp 9 16 PRT Mastadenovirus,serotype 2 9 Thr Val Ala Ser Val Ser Ile Phe Leu Arg Phe Asp Gln Asn GlyVal 1 5 10 15 10 14 PRT Mastadenovirus, serotype 5 10 Leu Asp Pro GluTyr Trp Asn Phe Arg Asn Gly Asp Leu Thr 1 5 10 11 13 PRT Mastadenovirus,serotype 2 11 Asn Ser Ser Leu Lys Lys His Tyr Trp Asn Phe Arg Asn 1 5 1012 42 DNA Mastadenovirus, serotype 5 12 cctaaactag gatccggcct tagttttgacagcatgggtg cc 42 13 37 DNA Mastadenovirus, serotype 5 13 ctgtgagtttgattaaggta ccgtgatctg tataagc 37 14 36 DNA Mastadenovirus, serotype 3 14ggtcttacat ttgactcttc catggctatt gcactg 36 15 35 DNA Mastadenovirus,serotype 3 15 caataaaaaa tgtggtacct tatttttgtt gtcag 35 16 51 DNA Homosapiens 16 tcgagaggca tatactttgg actcctgcta atacaccggc aatggggtat g 5117 51 DNA Homo sapiens 17 tcgacatacc ccattgccgg tgtattagca ggagtccaaagtatatgcct c 51 18 65 DNA Homo sapiens 18 tcgagagggc tatagttgggtttagggtgc aatggcttag gcggtatttt gtgaatgggt 60 cgagg 65 19 65 DNA Homosapiens 19 tcgacctcga cccattcaca aaataccgcc taagccattg caccctaaacccaactatag 60 ccctc 65 20 30 DNA Mastadenovirus, serotype 5 20ccatccgcac ccactatgat cacgttgttg 30 21 27 DNA Mastadenovirus, serotype 321 cttcatttct ttatcccccc atggcca 27 22 35 PRT Homo sapiens 22 Arg AlaIle Val Gly Phe Arg Val Gln Trp Leu Arg Arg Tyr Phe Val 1 5 10 15 AsnGly Ser Arg Lys Met Tyr Pro Arg Gly Asn His Trp Ala Val Gly 20 25 30 HisLeu Met 35 23 35 PRT Homo sapiens 23 Lys Met Tyr Pro Arg Gly Asn His TrpAla Val Gly His Leu Met Arg 1 5 10 15 Ala Ile Val Gly Phe Arg Val GlnTrp Leu Arg Arg Tyr Phe Val Asn 20 25 30 Gly Ser Arg 35 24 10 PRTArtificial Sequence Gastrin Releasing Peptide (GRP) 24 Gly Asn His TrpAla Val Gly His Leu Met 1 5 10 25 6 PRT Artificial Sequence Phagotope 25Gly Ile Ser His Thr Gly 1 5 26 6 PRT Artificial Sequence Phagotope 26Gly Ala Ser His Thr Val 1 5 27 6 PRT Artificial Sequence Phagotope 27Gln Ser Ala His Leu Ile 1 5 28 5 PRT Artificial Sequence Phagotope 28Leu Ala Pro Ile Ser 1 5 29 4 PRT Artificial Sequence Phagotope 29 ValAla Pro Ser 1 30 6 PRT Artificial Sequence Phagotope 30 Leu Ile Pro PheAsn Ser 1 5 31 4 PRT Artificial Sequence Phagotope 31 Tyr Trp Asn Phe 132 6 PRT Artificial Sequence Phagotope 32 Phe Trp Leu Ala Val Arg 1 5 336 PRT Artificial Sequence Phagotope 33 Trp Ala Leu Phe Arg Ser 1 5 34 5PRT Artificial Sequence Phagotope 34 Tyr Trp Asn Phe Arg 1 5 35 28 PRTArtificial Sequence Phagotope 35 Val Leu Ala Val Lys Gly Ser Leu Ala ProIle Ser Gly Thr Val Gln 1 5 10 15 Ser Ala His Leu Ile Ile Arg Phe AspGlu Asn Gly 20 25 36 6 PRT Artificial Sequence Phagotope 36 Val Phe ValLys Leu Pro 1 5 37 6 PRT Artificial Sequence Phagotope 37 Pro Asp ValAla Pro Ser 1 5 38 6 PRT Artificial Sequence Phagotope 38 Leu Ile ProPhe Asn Ser 1 5 39 6 PRT Artificial Sequence Phagotope 39 Leu Ser AsnGln Ser Gly 1 5 40 6 PRT Artificial Sequence Phagotope 40 Ser Gly ValGly Gln Ala 1 5 41 6 PRT Artificial Sequence Phagotope 41 Ser Val GlyAsp Tyr Gly 1 5 42 6 PRT Artificial Sequence Phagotope 42 Gly Ile SerHis Thr Gly 1 5 43 6 PRT Artificial Sequence Phagotope 43 Gly Ala SerHis Thr Val 1 5 44 6 PRT Artificial Sequence Phagotope 44 His Gly GlnTyr Arg Met 1 5 45 6 PRT Artificial Sequence Phagotope 45 Arg Arg IlePhe Arg Asp 1 5 46 17 PRT Artificial Sequence Phagotope 46 Asn Ser PheLeu Asp Pro Glu Tyr Trp Asn Phe Arg Asn Gly Asp Leu 1 5 10 15 Thr 47 6PRT Artificial Sequence Phagotope 47 Met Gln Pro Val Tyr Phe 1 5 48 6PRT Artificial Sequence Phagotope 48 Leu Gly Pro Val Asn Ser 1 5 49 6PRT Artificial Sequence Phagotope 49 Ala Leu Pro His Ile Val 1 5 50 6PRT Artificial Sequence Phagotope 50 Ala Pro His Glu Leu Arg 1 5 51 6PRT Artificial Sequence Phagotope 51 Met Asn Val Gly Ala His 1 5 52 6PRT Artificial Sequence Phagotope 52 Val Thr Ser Thr Tyr His 1 5 53 6PRT Artificial Sequence Phagotope 53 Leu Gln Lys Val His Arg 1 5 54 6PRT Artificial Sequence Phagotope 54 Asp Leu Trp Ser Val Leu 1 5 55 6PRT Artificial Sequence Phagotope 55 Phe Trp Leu Ala Val Arg 1 5 56 6PRT Artificial Sequence Phagotope 56 Trp Ala Leu Phe Arg Ser 1 5 57 6PRT Artificial Sequence Phagotope 57 Tyr Leu Gly Phe Phe Lys 1 5 58 6PRT Artificial Sequence Phagotope 58 Ile Ala Arg Leu Ile Ser 1 5 59 6PRT Artificial Sequence Phagotope 59 Arg Asn Tyr Thr Leu Thr 1 5 60 6PRT Artificial Sequence Phagotope 60 Arg Asp Ala Val Met Ile 1 5 61 6PRT Artificial Sequence Phagotope 61 Ser Arg Pro Thr Met Leu 1 5 62 6PRT Artificial Sequence Phagotope 62 Arg His Arg Met Leu Gln 1 5 63 6PRT Artificial Sequence Phagotope 63 Arg Arg His Trp Pro Phe 1 5 64 6PRT Artificial Sequence Phagotope 64 Trp Tyr Glu Trp Ile Gly 1 5 65 6PRT Artificial Sequence Phagotope 65 Trp Val Ile Trp Ser Ile 1 5 66 6PRT Artificial Sequence Phagotope 66 Ile Leu Trp Thr Pro Gly 1 5 67 6PRT Artificial Sequence Phagotope 67 Leu Gln Tyr Ser Leu Pro 1 5 68 6PRT Artificial Sequence Phagotope 68 Leu Leu Asp Phe Pro Ala 1 5 69 6PRT Artificial Sequence Phagotope 69 Leu Thr Pro Asn Thr Ile 1 5 70 6PRT Artificial Sequence Phagotope 70 Leu Gly Lys Ala Leu Pro 1 5 71 6PRT Artificial Sequence Phagotope 71 Ser Pro His Gly Ser Gly 1 5 72 6PRT Artificial Sequence Phagotope 72 Ala Pro Met Val Ala Leu 1 5 73 6PRT Artificial Sequence Phagotope 73 Thr Ala Ala Met Tyr Arg 1 5 74 6PRT Artificial Sequence Phagotope 74 Leu Phe Ile Ala Arg Leu 1 5 75 6PRT Artificial Sequence Phagotope 75 Tyr Leu Tyr Gly Arg Val 1 5 76 6PRT Artificial Sequence Phagotope 76 Ala Arg Val Ser Arg Ser 1 5 77 20PRT Artificial Sequence Phagotope 77 Arg His Ile Leu Trp Thr Pro Ala AsnThr Pro Ala Met Gly Tyr Leu 1 5 10 15 Ala Arg Val Ser 20 78 26 PRTArtificial Sequence Phagotope 78 Asn Ser His Pro Ile Gln Trp Asn Ala ProGln Pro Ser His Ile Ser 1 5 10 15 Lys Tyr Ile Leu Arg Trp Arg Pro LysAsn 20 25 79 25 PRT Artificial Sequence Phagotope 79 Val Lys Val Thr IleMet Trp Thr Pro Pro Glu Ser Ala Val Thr Gly 1 5 10 15 Tyr Arg Val AspVal Ile Pro Val Asn 20 25 80 25 PRT Artificial Sequence Phagotope 80 SerThr Val Leu Val Arg Trp Thr Pro Pro Arg Ala Gln Ile Thr Gly 1 5 10 15Tyr Arg Leu Thr Val Gly Leu Thr Arg 20 25 81 25 PRT Artificial SequencePhagotope 81 Asn Ser Leu Leu Val Ser Trp Gln Pro Pro Arg Ala Arg Ile ThrGly 1 5 10 15 Tyr Ile Ile Lys Tyr Glu Lys Pro Ser 20 25 82 6 PRTArtificial Sequence Phagotope 82 Ala Arg Ala Ile Val Gly 1 5 83 6 PRTArtificial Sequence Phagotope 83 Phe Val Trp Gly Leu Ser 1 5 84 6 PRTArtificial Sequence Phagotope 84 Phe Arg Val Gln Trp Leu 1 5 85 6 PRTArtificial Sequence Phagotope 85 Gln Val His Leu Phe Arg 1 5 86 6 PRTArtificial Sequence Phagotope 86 Val Gln Trp Phe Lys Pro 1 5 87 6 PRTArtificial Sequence Phagotope 87 Trp Ile Phe Leu Met Gln 1 5 88 6 PRTArtificial Sequence Phagotope 88 Arg Arg Tyr Phe Val Asn 1 5 89 6 PRTArtificial Sequence Phagotope 89 Tyr Phe Gly Ser Asn Ser 1 5 90 6 PRTArtificial Sequence Phagotope 90 Ala Tyr Gly Val Met Pro 1 5 91 6 PRTArtificial Sequence Phagotope 91 Leu Ala Pro Leu Gly Lys 1 5 92 6 PRTArtificial Sequence Phagotope 92 Ser Arg Leu Lys Met Gly 1 5 93 6 PRTArtificial Sequence Phagotope 93 His Met Glu Leu Leu Met 1 5 94 6 PRTArtificial Sequence Phagotope 94 His Ser Asn Gly Ser Arg 1 5 95 6 PRTArtificial Sequence Phagotope 95 Thr Arg Val Arg Thr Ser 1 5 96 6 PRTArtificial Sequence Phagotope 96 Arg Ser Glu Glu Thr Ile 1 5 97 24 PRTArtificial Sequence Phagotope 97 Ala Arg Ala Ile Val Gly Phe Arg Val GlnTrp Leu Arg Arg Tyr Phe 1 5 10 15 Val Asn Gly Ser Arg Glu Thr Ile 20 9825 PRT Artificial Sequence Phagotope 98 Leu Arg Ala Tyr Leu Glu Gly ThrCys Val Glu Trp Leu Arg Arg Tyr 1 5 10 15 Leu Glu Asn Gly Lys Glu ThrLeu Gln 20 25

1. Use of a polypeptide comprising an amino acid sequence homologous oridentical to at least 6 continuous amino acids of the sequence as shown:(a) in SEQ ID NO: 1 starting with the leucine residue at position 1 andending with the glutamine residue at position 25, (b) in SEQ ID NO: 2starting with the asparagine residue at position 1 and ending with theasparagine residue at position 26, (c) in SEQ ID NO: 3 starting with thevaline residue at position 1 and ending with the asparagine residue atposition 25, (d) in SEQ ID NO: 4 starting with the serine residue atposition 1 and ending with the arginine residue at position 25, and/or(e) in SEQ ID NO: 5 starting with the asparagine residue at position 1and ending with the serine residue at position 25; to allow orfacilitate the attachment of an adenovirus to a host cell and/or theentry of the said adenovirus into the said host cell.
 2. Use accordingto claim 1, characterized in that the polypeptide comprises an aminoacid sequence homologous or identical to at least 6 continuous aminoacids of the sequence as shown in SEQ ID NO: 1 starting with the leucineresidue at position 1 and ending with the glutamine residue at position25.
 3. Use according to claim 2, characterized in that the polypeptidecomprises an amino acid sequence homologous or identical to all or partof an antigen of the class I major histocompatibility complex (MHC-I)and, preferably, of the heavy chain of the said MHC-I.
 4. Use accordingto claim 3, characterized in that the polypeptide comprises an aminoacid sequence homologous or identical to all or part of the α2 domain ofthe said heavy chain.
 5. Use according to claim 3 or 4, characterized inthat the polypeptide comprises an amino acid sequence homologous oridentical to all or part of an HLA A, B, C, D, E or F antigen.
 6. Useaccording to claim 1, characterized in that the polypeptide comprises anamino acid sequence homologous or identical to at least 6 continuousamino acids of the sequence as shown: in SEQ ID NO: 2 starting with theasparagine residue at position 1 and ending with the asparagine residueat position 26, in SEQ ID NO: 3 starting with the valine residue atposition 1 and ending with the asparagine residue at position 25, in SEQID NO: 4 starting with the serine residue at position 1 and ending withthe arginine residue at position 25, and/or in SEQ ID NO: 5 startingwith the asparagine residue at position 1 and ending with the serineresidue at position
 25. 7. Use according to claim 6, characterized inthat the said polypeptide comprises an amino acid sequence homologous oridentical to all or part of fibronectin.
 8. Use according to claim 7,characterized in that the said polypeptide comprises an amino acidsequence homologous or identical to all or part of at least one type IIImodule of fibronectin.
 9. Use according to one of claims 1 to 8,characterized in that the said adenovirus is of serotype C.
 10. Useaccording to claim 9, characterized in that the said adenovirus is ofserotype 2 or
 5. 11. Cell capable of expressing a polypeptide as definedaccording to one of claims 1 to
 10. 12. Cell according to claim 11,characterized in that the said cell overexpresses the said polypeptide.13. Use of a cell according to claim 11 or 12, to allow or facilitatethe attachment of an adenovirus to the said cell and/or the entry of thesaid adenovirus into the said cell.
 14. Use of a ligand to influence theattachment of an adenovirus to a host cell and/or the entry of the saidadenovirus into the said host cell which are mediated by a polypeptideas defined in one of claims 1 to
 10. 15. Use of a ligand according toclaim 14, to negatively influence the attachment of an adenovirus to thesaid host cell and/or the entry of the said adenovirus into the saidhost cell which are mediated by a polypeptide as defined according toone of claims 1 to 5 and 9 and
 10. 16. Use of a ligand according toclaim 15, characterized in that the said ligand comprises an amino acidsequence homologous or identical to at least 6 continuous amino acidscontained in the sequence as shown in SEQ ID NO: 6 starting with thearginine residue at position 1 and ending with the arginine residue atposition
 20. 17. Use of a ligand according to claim 14, to positivelyinfluence the attachment of an adenovirus to the said host cell and/orthe entry of the said adenovirus into the said host cell which aremediated by a polypeptide as defined according to one of claims 1 and 6to
 10. 18. Use of a ligand according to claim 17, characterized in thatthe said ligand comprises an amino acid sequence homologous or identicalto at least 6 continuous amino acids contained in the sequence as shownin SEQ ID NO: 7 starting with the arginine residue at position 1 andending with the serine residue at position
 20. 19. Use of a ligandaccording to one of claims 14 to 18, characterized in that the saidligand has a dissociation constant (kd) with respect to the saidadenovirus of 0.01 to 100 nM, and in particular of 0.1 to 50 nM. 20.Ligand as defined in claim 16, 18 or
 19. 21. Use of a ligand accordingto claim 14, characterized in that the said ligand is of adenoviralorigin.
 22. Use according to claim 21, characterized in that the saidligand is derived from the fiber of an adenovirus and, in particular,from the head domain.
 23. Use according to claim 22 to interact with apolypeptide as defined according to one of claims 1 to 5 and 9 and 10,characterized in that the said ligand is derived from the fiber of aserotype 5 adenovirus and comprises an amino acid sequence homologous oridentical to at least 6 continuous amino acids of the sequence as shownin SEQ ID NO: 8 starting with the leucine residue at position 1 andending with the aspartic acid residue at position
 18. 24. Use accordingto claim 22 to interact with a polypeptide as defined according to oneof claims 1 to 5 and 9 and 10, characterized in that the said ligand isderived from the fiber of a serotype 2 adenovirus and comprises an aminoacid sequence homologous or identical to at least 6 continuous aminoacids of the sequence as shown in SEQ ID NO: 9 starting with thethreonine residue at position 1 and ending with the valine residue atposition
 16. 25. Use according to claim 22 to interact with apolypeptide as defined according to one of claims 1 and 6 to 10,characterized in that the said ligand is derived from the fiber of aserotype 5 adenovirus and comprises an amino acid sequence homologous oridentical to at least 6 continuous amino acids of the sequence as shownin SEQ ID NO: 10 starting with the leucine residue at position 1 andending with the threonine residue at position
 14. 26. Use according toclaim 22 to interact with a polypeptide as defined according to one ofclaims 1 and 6 to 10, characterized in that the said ligand is derivedfrom the fiber of a serotype 2 adenovirus and comprises an amino acidsequence homologous or identical to at least 6 continuous amino acids ofthe sequence as shown in SEQ ID NO: 11 starting with the asparagineresidue at position 1 and ending with the asparagine residue at position13.
 27. Use of a ligand according to one of claims 14 to 26, for thepreparation of a medicament intended to inhibit or reduce an infectionby an adenovirus.
 28. Use of a ligand according to one of claims 14 to26, for the preparation of a medicament intended to promote orfacilitate an infection by an adenovirus, and in particular arecombinant adenovirus.
 29. Method for selecting or identifying acellular receptor for a virus in an appropriate sample, comprising thefollowing steps: (a) immobilization, onto an inert support, of a reagentof viral origin comprising all or part of a surface protein of the saidvirus which determines the attachment of the said virus to the saidcellular receptor, (b) incubation with the said sample for a determinedtime, (c) elution of the sample retained in step (b) with all or part ofan antibody directed against the said reagent of viral origin, and (d)analysis of the sample eluted in step (c).
 30. Method for selecting oridentifying the part of a viral protein which determines the attachmentof a virus to a cellular receptor in an appropriate sample, comprisingthe following steps: (a) immobilization, onto an inert support, of allor part of an antibody directed against the said viral protein, (b)incubation with the said sample for a determined time, (c) elution ofthe sample retained in step (b) with a reagent of viral origincomprising all or part of the said viral protein, and (d) analysis ofthe sample eluted in step (c).
 31. Method according to claim 29 or 30,characterized in that the said sample is a peptide library or a phagelibrary expressing peptide motifs.
 32. Method according to one of claims29 to 31, characterized in that the said virus is an adenovirus, inparticular of serotype C, the reagent of viral origin comprises to allor part of the fiber and, in particular, of the head of an adenovirusand the antibody is an inhibitor of the attachment of the virus to thesurface of the host cell.
 33. Use of a bifunctional ligand for targetingan adenovirus to a cell surface protein other than the natural cellularreceptor for the said adenovirus, the said bifunctional ligandcomprising a first ligand part capable of interacting with the fiber ofthe said adenovirus, a second ligand part capable of interacting withthe said cell surface protein and, optionally, a spacer between the saidfirst and second ligand parts; the said first ligand part having thecharacteristics of the ligand as defined in claims 14 to
 20. 34. Useaccording to claim 33, according to which the said first ligand partcomprises an amino acid sequence homologous or identical to at least 6continuous amino acids contained in SEQ ID NO: 6 and more particularlyhas an amino acid sequence identical to SEQ ID NO: 6 starting with thearginine residue at position 1 and ending with the arginine residue atposition
 20. 35. Use according to claim 33 or 34, according to which thesaid bifunctional ligand comprises an amino acid sequence homologous oridentical to all or part of the sequence as shown: (i) in SEQ ID NO: 22starting at the arganine residue at position 1 and ending with themethionine residue at position 35, or (ii) in SEQ ID NO: 23 starting atthe lysine residue at position 1 and ending with the arginine residue atposition
 35. 36. Bifunctional ligand as defined in one of claims 33 to35.
 37. Cell carrying at its surface a bifunctional ligand according toclaim 36.